Methods for promoting growth of bone, ligament, and cartilage

ABSTRACT

Methods for promoting growth of bone, ligament, or cartilage in a mammal are disclosed. The methods comprise administering to said mammal a composition comprising a pharmacologically effective amount of a zvegf3 protein in combination with a pharmaceutically acceptable delivery vehicle. Also disclosed are methods for promoting proliferation or differentiation of osteoblasts, osteoclasts, chondrocytes, or bone marrow stem cells comprising culturing the cells in an effective amount of a zvegf3 protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/664,432,filed Sep. 19, 2003, now U.S. Pat. No. 7,491,384 incorporated herein byreference, and which is a division of application Ser. No. 09/823,033,filed Mar. 29, 2001, incorporated herein by reference, now U.S. Pat. No.6,663,870, which claims the benefit of provisional application Ser. No.60/193,723, filed Mar. 31, 2000 and which is a continuation-in-part ofapplication Ser. No. 09/457,066, filed Dec. 7, 1999, now U.S. Pat. No.6,432,673, which claims the benefit of provisional applications Ser. No.60/165,255, filed Nov. 12, 1999, Ser. No. 60/161,653, filed Oct. 21,1999, Ser. No. 60/142,576, filed Jul. 6, 1999, and Ser. No. 60/111,173,filed Dec. 7, 1998.

BACKGROUND OF THE INVENTION

Bone remodeling is the dynamic process by which tissue mass and skeletalarchitecture are maintained. The process is a balance between boneresorption and bone formation, with two cell types, the osteoclast andosteoblast, thought to be the major players. Osteoblasts synthesize anddeposit new bone into cavities that are excavated by osteoclasts. Theactivities of osteoblasts and osteoclasts are regulated by many factors,systemic and local, including growth factors.

Many of the proteins that influence the proliferation, differentiation,and activity of osteoblasts, osteoclasts, and their precursors alsoaffect these processes in chondrocytes, the cells responsible forcartilage formation (chondrogenesis). These proteins includeplatelet-derived growth factor (PDGF), insulin-like growth factor (IGF),basic fibroblast growth factor (bFGF), transforming growth factor beta(TGF-β), bone morphogenetic proteins (BMPs), and cartilage-derivedgrowth factor (CDGF).

The exact mode by which PDGF affects the growth of osteoblasts is notyet clearly understood, however, this growth factor is generallybelieved to play a key role in the regulation of both normal skeletalremodeling and fracture repair. Biologically active PDGF is found as ahomodimer or a heterodimer of the component A and B chains. In vitrostudies have shown PDGF to be mitogenic for osteoblasts (Abdennagy etal., Cell Biol. Internat. Rep. 16(3):235-247, 1992). Mitogenic activityas well as chemotactic activities associated with PDGF have beendemonstrated when the growth factor is added to normal osteoblast-likecells (Tuskamota et al., Biochem. Biophys. Res. Comm. 175(3):745-747,1991) and primary osteoblast cultures (Centrella et al. Endocrinol.125(1):13-19, 1989). Recent studies have demonstrated that theosteoblast produces the AA isoform of PDGF (Zhang et al., Am. J.Physiol. 261:c348-c354, 1991).

PDGF has been shown to be useful for promoting the repair of both softand hard tissues. For example, PDGF has been shown to promote theregeneration of bone and ligament in patients suffering from periodontaldisease (Howell et al., J. Periodontol. 68:1186-1193, 1997). Asdisclosed in U.S. Pat. No. 5,533,836, PDGF stimulates the growth ofosteoblasts, and this activity is enhanced in the presence of vitamin D.PDGF has also been shown to promote the healing of gastrointestinalulcers (U.S. Pat. No. 5,234,908) and dermal ulcers (Robson et al.,Lancet 339:23-25, 1992; Steed et al., J. Vasc. Surg. 21:71-81, 1995).The use of PDGF for stimulating chondrocyte proliferation andregenerating cartilage is disclosed in U.S. Pat. No. 6,001,352.

A PDGF homolog known as “zvegf3” was recently identified (U.S. patentapplication Ser. No. 09/457,066). This protein has also been designated“VEGF-R” (WIPO Publication WO 99/37671). Zvegf3/VEGF-R is a multi-domainprotein with significant homology to the PDGF/VEGF family of growthfactors. WO 99/37671 discloses that VEGF-R is an angiogenic factor.

Despite the increasing knowledge of the role of growth factors in tissuegrowth and repair, there remains a need in the art for materials andmethods for promoting the growth of bone, ligament, and cartilage. Therealso remains a need the art for materials and methods for modulating theproliferation and differentiation of cells in vitro and in vivo.

DESCRIPTION OF THE INVENTION

The present invention provides a method for promoting growth of bone,ligament, or cartilage in a mammal comprising administering to saidmammal a composition comprising a pharmacologically effective amount ofa dimeric protein comprising residues 235-345 of SEQ ID NO:2 or SEQ IDNO:4 and a pharmaceutically acceptable delivery vehicle. Within certainembodiments of the invention the delivery vehicle is powdered bone,tricalcium phosphate, hydroxyapatite, polymethacrylate, a biodegradablepolyester, an aqueous polymeric gel, or a fibrin sealant. Within anotherembodiment of the invention the composition is locally administered at asite of a bony defect, such as a fracture, bone graft site, implantsite, or periodontal pocket. Within another embodiment of the invention,the composition is administered systemically. Within a furtherembodiment of the invention, the zvegf3 protein is covalently linked toa bone-targeting agent. Within a further embodiment of the invention,the composition is locally administered at a joint. The composition mayfurther comprise a protein selected from the group consisting ofinsulin-like growth factor 1, platelet-derived growth factor, epidermalgrowth factor, transforming growth factor-alpha, transforming growthfactor-beta, a bone morphogenetic protein, parathyroid hormone,osteoprotegerin, a fibroblast growth factor, and a protein comprisingresidues 258-370 of SEQ ID NO:5 (a zvegf4 protein). Within anotherembodiment of the invention, the protein is a homodimer. Within arelated embodiment, the protein comprises a first polypeptide chaindisulfide bonded to a second polypeptide chain, each of the chainsconsisting of residues X-345 of SEQ ID NO:2, wherein X is an integerfrom 226 to 235, inclusive.

The invention also provides a method for promoting growth of bone,ligament, or cartilage in a mammal comprising administering to saidmammal a composition comprising a pharmacologically effective amount ofa dimeric protein comprising a first polypeptide chain disulfide bondedto a second polypeptide chain, each of the chains comprising of residues235-345 of SEQ ID NO:2 or SEQ ID NO:4, and a pharmaceutically acceptabledelivery vehicle. Within certain embodiments, each of the chainsconsists of residues X-345 of SEQ ID NO:2, wherein X is an integer from226 to 235, inclusive. Within other embodiments, each of the chainsconsists of residues X-345 of SEQ ID NO:2, wherein X is an integer from15 to 20, inclusive.

The invention also provides a method for promoting proliferation ordifferentiation of cells comprising culturing the cells in an effectiveamount of a dimeric protein comprising residues 235-345 of SEQ ID NO:2or SEQ ID NO:4, wherein the cells are osteoblasts, osteoclasts,chondrocytes, or bone marrow stem cells. Within one embodiment the cellsare bone marrow stem cells, and the method comprises harvesting the bonemarrow stem cells from a patient prior to culturing. Within otherembodiments the method further comprises the step of recoveringosteoblasts, osteoclasts, or chrodrocytes from the cultured cells.Within additional embodiments the protein comprises a first polypeptidechain disulfide bonded to a second polypeptide chain, each of the chainsconsisting of residues X-345 of SEQ ID NO:2, wherein X is an integerfrom 226 to 235, inclusive.

The invention also provides a method for promoting cartilage growthcomprising the steps of (a) culturing chondrocytes ex vivo in thepresence of a dimeric protein comprising residues 235-345 of SEQ ID NO:2or SEQ ID NO:4 under conditions wherein the chondrocytes proliferate,and (b) placing the cultured chondrocytes into a mammal where cartilageis to be grown. Within one embodiment the chondrocytes are placed intothe mammal in association with a biodegradable matrix having sufficientporosity to permit cell ingrowth. Within a related embodiment the matrixcomprises a protein selected from the group consisting of insulin-likegrowth factor 1, platelet-derived growth factor, epidermal growthfactor, transforming growth factor-alpha, transforming growthfactor-beta, a bone morphogenetic protein, parathyroid hormone, afibroblast growth factor, a protein comprising residues 258-370 of SEQID NO:5, and a dimeric protein comprising residues 235-345 of SEQ IDNO:2 or SEQ ID NO:4. Within other embodiments the protein comprises afirst polypeptide chain disulfide bonded to a second polypeptide chain,each of the chains consisting of residues X-345 of SEQ ID NO:2, whereinX is an integer from 226 to 235, inclusive.

The invention further provides a method for stimulating proliferation ofosteoblasts or chondrocytes in a mammal comprising administering to themammal a composition comprising a pharmacologically effective amount ofa dimeric protein comprising residues 235-345 of SEQ ID NO:2 or SEQ IDNO:4 in combination with a pharmaceutically acceptable delivery vehicle.Within certain embodiments of the invention the delivery vehicle ispowdered bone, tricalcium phosphate, hydroxyapatite, polymethacrylate, abiodegradable polyester, an aqueous polymeric gel, or a fibrin sealant.Within another embodiment the composition is locally administered at asite of a bony defect, such as a fracture, bone graft site, implantsite, or periodontal pocket. Within another embodiment the compositionis administered systemically. Within a further embodiment the zvegf3protein is covalently linked to a bone-targeting agent. Within anadditional embodiment the composition is locally administered at ajoint. Within other embodiments the composition further comprises aprotein selected from the group consisting of insulin-like growth factor1, platelet-derived growth factor, epidermal growth factor, transforminggrowth factor-alpha, transforming growth factor-beta, a bonemorphogenetic protein, parathyroid hormone, osteoprotegerin, afibroblast growth factor, and a protein comprising residues 258-370 ofSEQ ID NO:5. Within additional embodiments the protein comprises a firstpolypeptide chain disulfide bonded to a second polypeptide chain, eachof the chains consisting of residues X-345 of SEQ ID NO:2, wherein X isan integer from 226 to 235, inclusive.

These and other aspects of the invention will become evident uponreference to the following detailed disclosure and the accompanyingdrawings. Within the drawings:

FIG. 1 illustrates an alignment of representative human (SEQ ID NO:2)and mouse (SEQ ID NO:4) zvegf3 amino acid sequences.

FIGS. 2A-2G are a Hopp/Woods hydrophilicity profile of the amino acidsequence shown in SEQ ID NO:2. The profile is based on a slidingsix-residue window. Buried G, S, and T residues and exposed H, Y, and Wresidues were ignored. These residues are indicated in the figure bylower case letters.

As used herein, the term “bony defect” denotes a defect or void in abone where restoration of the bone is desirable. Bony defects may arisefrom injury, surgery, tumor removal, ulceration, infection, or othercauses, and include congenital defects. Examples of bony defects includefractures, voids resulting from tumor removal, and bone loss resultingfrom periodontal disease.

The terms “locally administered” and “local administration” are used todescribe the application of a pharmaceutical agent at the intended siteof action. Examples of local administration include, without limitation,injection into a joint space, implantation of a solid or semi-solidmatrix, and direct application at a surgical site or wound. Localadministration does not preclude the transmission of minor amounts ofthe agent to other parts of the body, such as by diffusion orcirculation.

The term “zvegf3 protein” is used herein to denote proteins comprisingthe growth factor domain of a zvegf3 polypeptide (e.g., residues 235-345of human zvegf3 (SEQ ID NO:2) or mouse zvegf3 (SEQ ID NO:4)), whereinsaid protein is mitogenic for cells expressing cell-surface PDGFα-receptor subunit. Zvegf3 has been found to bind to the αα and αβisoforms of PDGF receptor. Experimental evidence indicates thatbiologically active zvegf3 is a dimeric protein. Zvegf3 proteins includehomodimers and heterodimers as disclosed below. Using methods known inthe art, zvegf3 proteins can be prepared in a variety of forms,including glycosylated or non-glycosylated; pegylated or non-pegylated;with or without an initial methionine residues; and as fusion proteinsas disclosed in more detail below.

The present invention provides methods for promoting the growth of bone,connective tissue (including ligament, tendon, and cartilage), andrelated cell types using zvegf3 proteins. Zvegf3 is a protein that isstructurally related to platelet-derived growth factor (PDGF) and thevascular endothelial growth factors (VEGF). The zvegf3 polypeptide chaincomprises a growth factor domain and a CUB domain. The growth factordomain is characterized by an arrangement of cysteine residues and betastrands that is characteristic of the “cystine knot” structure of thePDGF family. The CUB domain shows sequence homology to CUB domains inthe neuropilins (Takagi et al., Neuron 7:295-307, 1991; Soker et al.,ibid.), human bone morphogenetic protein-1 (Wozney et al., Science242:1528-1534, 1988), porcine seminal plasma protein and bovine acidicseminal fluid protein (Romero et al., Nat. Struct. Biol. 4:783-788,1997), and X. laevis tolloid-like protein (Lin et al., Dev. GrowthDiffer. 39:43-51, 1997).

Structural predictions based on the zvegf3 sequence and its homology toother growth factors suggests that the polypeptide can formhomomultimers or heteromultimers having growth factor activity, i.e.,modulating one or more of cell proliferation, migration,differentiation, and metabolism. While not wishing to be bound bytheory, the similarity of zvegf3 to other members of the PDGF/VEGFfamily suggests that zvegf3 may also form heteromultimers with othermembers of the family, including VEGF, VEGF-B, VEGF-C, VEGF-D, zvegf4(SEQ ID NO:5), P1GF (Maglione et al., Proc. Natl. Acad. Sci. USA88:9267-9271, 1991), PDGF-A (Murray et al., U.S. Pat. No. 4,899,919;Heldin et al., U.S. Pat. No. 5,219,759), or PDGF-B (Chiu et al., Cell37:123-129, 1984; Johnsson et al., EMBO J. 3:921-928, 1984).

A representative human zvegf3 polypeptide sequence is shown in SEQ IDNO:2, and a representative mouse zvegf3 polypeptide sequence is shown inSEQ ID NO:4. DNAs encoding these polypeptides are shown in SEQ ID NOS:1and 3, respectively. An alignment of the mouse and human polypeptidesequences is shown in FIG. 1. Analysis of the amino acid sequence shownin SEQ ID NO:2 indicates that residues 1 to 14 form a secretory peptide.The CUB domain extends from residue 46 to residue 163. A propeptide-likesequence extends from residue 164 to residue 234, and includes twopotential cleavage sites at its carboxyl terminus, a dibasic site atresidues 231-232 and a target site for furin or a furin-like protease atresidues 231-234. The growth factor domain extends from residue 235 toresidue 345. Those skilled in the art will recognize that domainboundaries are somewhat imprecise and can be expected to vary by up to±5 residues from the specified positions. Potential proteolytic cleavagesites occur at residues 232 and 234. Processing of recombinant zvegf3produced in BHK cells has been found to occur between residues 225 and226. Signal peptide cleavage is predicted to occur after residue 14 (±3residues). This analysis suggests that the zvegf3 polypeptide chain maybe cleaved to produce a plurality of monomeric species as shown inTable 1. Cleavage after Arg-234 is expected to result in subsequentremoval of residues 231-234, with possible conversion of Gly-230 to anamide. Cleavage after Lys-232 is expected to result in subsequentremoval of residue 231, again with possible conversion of Gly-230 to anamide. In addition, it may be advantageous to include up to sevenresidues of the interdomain region at the carboxyl terminus of the CUBdomain. The interdomain region can be truncated at its amino terminus bya like amount. See Table 1. Corresponding domains in mouse and othernon-human zvegf3s can be determined by those of ordinary skill in theart from sequence alignments.

TABLE 1 Monomer Residues (SEQ ID NO:2) Cub domain 15-163 46-163 15-17046-170 CUB domain + interdomain region 15-234 46-234 15-229 amide 15-230Cub domain + interdomain region + growth 15-345 factor domain 46-345Growth factor domain 235-345 226-345 Growth factor domain + interdomainregion 164-345 171-345

Zvegf3 can thus be prepared in a variety of multimeric forms comprisinga zvegf3 polypeptide as disclosed above. These zvegf3 polypeptidesinclude zvegf3₁₅₋₂₃₄, zvegf3₄₆₋₂₃₄, zvegf3₁₅₋₂₂₉ amide, zvegf3₁₅₋₂₃₀,zvegf3₁₅₋₃₄₅, zvegf3₄₆₋₃₄₅, and zvegf3₂₃₅₋₃₄₅. Variants and derivativesof these polypeptides can also be prepared as disclosed herein.

Zvegf3 proteins can be prepared as fusion proteins comprising amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, an affinity tag, or a targetting polypeptide. For example, azvegf3 protein can be prepared as a fusion with an affinity tag tofacilitate purification. In principal, any peptide or protein for whichan antibody or other specific binding agent is available can be used asan affinity tag. Affinity tags include, for example, a poly-histidinetract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith andJohnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al.,Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P, FLAG™ peptide(Hopp et al., Biotechnology 6:1204-1210, 1988), streptavidin bindingpeptide, maltose binding protein (Guan et al., Gene 67:21-30, 1987),cellulose binding protein, thioredoxin, ubiquitin, T7 polymerase, orother antigenic epitope or binding domain. Fusion of zvegf3 to, forexample, maltose binding protein or glutatione S transferase, can beused to improve yield in bacterial expression systems. In theseinstances the non-zvegf3 portion of the fusion protein ordinarily willbe removed prior to use. Separation of the zvegf3 and non-zvegf3portions of the fusion protein is facilitated by providing a specificcleavage site between the two portions. Such methods are well known inthe art. Zvegf3 can also be fused to a targetting peptide, such as anantibody (including polyclonal antibodies, monoclonal antibodies,antigen-binding fragments thereof such as F(ab′)₂ and Fab fragments,single chain antibodies, and the like), calcitonin, or other peptidicmoiety that binds to bone or connective tissue.

Variations can be made in the zvegf3 amino acid sequences shown in SEQID NO:2 and SEQ ID NO:4. Such variations include amino acidsubstitutions, deletions, and insertions. Amino acid sequence changesare made in zvegf3 polypeptides so as to minimize disruption of higherorder structure essential to biological activity. In general,conservative amino acid changes are preferred. Changes in amino acidresidues will be made so as not to disrupt the cystine knot and “bowtie” arrangement of loops in the growth factor domain that ischaracteristic of the protein family. Conserved motifs will also bemaintained. The effects of amino acid sequence changes can be predictedby computer modeling as disclosed above or determined by analysis ofcrystal structure (see, e.g., Lapthorn et al., Nature 369:455, 1994). Ahydrophobicity profile of SEQ ID NO:2 is shown in FIGS. 2A-2G. Thoseskilled in the art will recognize that this hydrophobicity will be takeninto account when designing alterations in the amino acid sequence of azvegf3 polypeptide, so as not to disrupt the overall profile. Additionalguidance in selecting amino acid substitutions is provided by thealignment of mouse and human zvegf3 sequences shown in FIG. 1. The aminoacid sequence is highly conserved between mouse and human zvegf3s, withan overall amino acid sequence identity of 87%. The secretory peptide,CUB domain, inter-domain, and growth factor domain have 82%, 92%, 79%and 94% amino acid identity, respectively.

It is preferred that the sequence of zvegf3 polypeptide be at least 95%identical to the corresponding region of SEQ ID NO:2 or SEQ ID NO:4.Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986, andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.). Thepercent identity is then calculated as:

$\frac{{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{identical}\mspace{14mu}{matches}}{\begin{matrix}\left\lbrack {{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{longer}\mspace{14mu}{sequence}\mspace{14mu}{plus}\mspace{14mu}{the}} \right. \\{{number}\mspace{14mu}{of}\mspace{14mu}{gaps}\mspace{14mu}{introduced}\mspace{14mu}{into}\mspace{14mu}{the}\mspace{14mu}{longer}} \\\left. {{sequence}\mspace{14mu}{in}\mspace{14mu}{order}\mspace{14mu}{to}\mspace{14mu}{align}\mspace{14mu}{the}\mspace{14mu}{two}\mspace{14mu}{sequences}} \right\rbrack\end{matrix}} \times 100$

Zvegf3 proteins can also comprise non-naturally occurring amino acidresidues. Non-naturally occurring amino acids include, withoutlimitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occurring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is carried out in a cell-freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Methods Enzymol 202:301, 1991; Chung et al., Science259:806-809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145-10149, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-19998, 1996). Within a third method, E. coli cells arecultured in the absence of a natural amino acid that is to be replaced(e.g., phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

Essential amino acids in zvegf3 proteins can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, Science 244,1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502,1991). Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Other methods that canbe used include phage display (e.g., Lowman et al., Biochem.30:10832-10837, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPOPublication WO 92/06204), region-directed mutagenesis (Derbyshire etal., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988), and DNA shufflingas disclosed by Stemmer (Nature 370:389-391, 1994) and Stemmer (Proc.Natl. Acad. Sci. USA 91:10747-10751, 1994). The resultant mutantmolecules are tested for mitogenic activity or other properties (e.g.,receptor binding) to identify amino acid residues that are critical tothe activity of the molecule. Mutagenesis can be combined with highvolume or high-throughput screening methods to detect biologicalactivity of zvegf3 variant polypeptides, in particular biologicalactivity in modulating cell proliferation or cell differentiation. Forexample, mitogenesis assays that measure dye incorporation or³H-thymidine incorporation can be carried out on large numbers ofsamples.

Using the methods discussed above, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptides that are homologous tothe zvegf3 polypeptides disclosed above in Table 1 and retain thebiological properties of the wild-type protein.

Zvegf3 proteins for use within the present invention, includingfull-length polypeptides, biologically active fragments, and fusionproteins, can be produced in genetically engineered host cells accordingto conventional techniques. Suitable host cells are those cell typesthat can be transformed or transfected with exogenous DNA and grown inculture, and include bacteria, fungal cells, and cultured highereukaryotic cells (including cultured cells of multicellular organisms).Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al.,eds., Current Protocols in Molecular Biology, Green and Wiley and Sons,NY, 1993.

In general, a DNA sequence encoding a zvegf3 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a zvegf3 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of zvegf3, or may be derivedfrom another secreted protein (e.g., t-PA; see, U.S. Pat. No. 5,641,655)or synthesized de novo. The secretory signal sequence is operably linkedto the zvegf3 DNA sequence, i.e., the two sequences are joined in thecorrect reading frame and positioned to direct the newly synthesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe polypeptide of interest, although certain signal sequences may bepositioned elsewhere in the DNA sequence of interest (see, e.g., Welchet al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.5,143,830).

Expression of zvegf3 polypeptides via a host cell secretory pathway isexpected to result in the production of multimeric proteins. As notedabove, such multimers include both homomultimers and heteromultimers,the latter including proteins comprising only zvegf3 polypeptides andproteins including zvegf3 and heterologous polypeptides. For example, aheteromultimer comprising a zvegf3 polypeptide and a polypeptide from arelated family member (e.g., VEGF, VEGF-B, VEGF-C, VEGF-D, zvegf4, P1GF,PDGF-A, or PDGF-B) can be produced by co-expression of the twopolypeptides in a host cell. Sequences encoding these other familymembers are known. See, for example, Dvorak et al, ibid.; Olofsson etal, ibid.; Hayward et al., ibid.; Joukov et al., ibid.; Oliviero et al.,ibid.; Achen et al., ibid.; Maglione et al., ibid.; Heldin et al., U.S.Pat. No. 5,219,759; and Johnsson et al., ibid. If a mixture of proteinsresults from expression, individual species are isolated by conventionalmethods. Monomers, dimers, and higher order multimers are separated by,for example, size exclusion chromatography. Heteromultimers can beseparated from homomultimers by immunoaffinity chromatography usingantibodies specific for individual dimers or by sequentialimmunoaffinity steps using antibodies specific for individual componentpolypeptides. See, in general, U.S. Pat. No. 5,094,941. Multimers mayalso be assembled in vitro upon incubation of component polypeptidesunder suitable conditions. In general, in vitro assembly will includeincubating the protein mixture under denaturing and reducing conditionsfollowed by refolding and reoxidation of the polypeptides to fromhomodimers and heterodimers. Recovery and assembly of proteins expressedin bacterial cells is disclosed below.

Zvegf3 proteins can be produced in eukaryotic host cells, includingfungal cells (e.g., Saccharomyces cerevisiae, Pichia methanolica, andPichia pastoris), mammalian cells, plant cells, and insect cellsaccording to conventional methods. See, for example, Kawasaki, U.S. Pat.No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S.Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; Murray etal., U.S. Pat. No. 4,845,075; Gleeson et al., J. Gen. Microbiol132:3459-3465, 1986; Cregg, U.S. Pat. No. 4,882,279; Raymond et al.,Yeast 14:11-23, 1998; McKnight et al., U.S. Pat. No. 4,935,349; Suminoet al., U.S. Pat. No. 5,162,228; Lambowitz, U.S. Pat. No. 4,486,533;Raymond et al., 5,854,039; Raymond, U.S. Pat. Nos. 5,716,808, 5,736,383,and 5,888,768; Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al.,U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821;Foster et al., U.S. Pat. No. 4,959,318; Mulvihill et al., U.S. Pat. No.5,648,254; Moore et al., U.S. Pat. No. 5,622,839; Kuestner et al., U.S.Pat. No. 6,008,322; Sinkar et al., J. Biosci. (Bangalore) 11:47-58,1987; Luckow et al., J. Virol. 67:4566-4579, 1993; Hill-Perkins andPossee, J. Gen. Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol75:1551-1556, 1994; and Chazenbalk and Rapoport, J. Biol. Chem.270:1543-1549, 1995. Suitable host strain and cell lines are known inthe art and available from public depositories such as the American TypeCulture Collection, Manassas, Va., USA. Suitable cultured mammaliancells include the COS-1 (ATCC® No. CRL 1650), COS-7 (ATCC® No. CRL1651), BHK (ATCC® No. CRL 1632), BHK 570 (ATCC® No. CRL 10314), 293(ATCC® No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) andChinese hamster ovary (e.g. CHO-K1; ATCC® No. CCL 61) cell lines.Expression vectors for use in mammalian cells include pZP-1 and pZP-9,which have been deposited with the American Type Culture Collection,Manassas, Va., USA under accession numbers 98669 and 98668,respectively. Cells, expression vectors, expression kits, and othermaterials are available from commercial suppliers.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera can also be used for production ofzvegf3 proteins. Techniques for transforming these hosts and expressingforeign DNA sequences cloned therein are well known in the art (see,e.g., Sambrook et al., ibid.). When expressing a zvegf3 polypeptide inbacteria such as E. coli, the polypeptide may be retained in thecytoplasm, typically as insoluble granules, or may be directed to theperiplasmic space by a bacterial secretion sequence. In the former case,the cells are lysed, and the granules are recovered and denatured using,for example, guanidine isothiocyanate or urea. The denatured polypeptidecan then be refolded and dimerized by diluting the denaturant, such asby dialysis against a solution of urea and a combination of reduced andoxidized glutathione, followed by dialysis against a buffered salinesolution. In the alternative, the protein may be recovered from thecytoplasm in soluble form and isolated without the use of denaturants.The protein is recovered from the cell as an aqueous extract in, forexample, phosphate buffered saline. To capture the protein of interest,the extract is applied directly to a chromatographic medium, such as animmobilized antibody or heparin-SEPHAROSE™ column. Secreted polypeptidescan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

Zvegf3 polypeptides or fragments thereof can also be prepared throughchemical synthesis according to methods known in the art, includingexclusive solid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. See, for example,Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid PhasePeptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, Ill.,1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford,1989.

Covalent complexes can also be made by isolating the desired componentpolypeptides and combining them in vitro. Covalent complexes that can beprepared in this manner include homodimers of zvegf3 polypeptides,heterodimers of two different zvegf3 polypeptides, and heterodimers of azvegf3 polypeptide and a polypeptide from another family member of theVEGF/PDGF family of proteins. The two polypeptides are mixed togetherunder denaturing and reducing conditions, followed by renaturation ofthe proteins by removal of the denaturants. Removal can be done by, forexample, dialysis or size exclusion chromatography to provide for bufferexchange. When combining two different polypeptides, the resultingrenaturated proteins may form homodimers of the individual components aswell as heterodimers of the two polypeptide components. See, Cao et al.,J. Biol. Chem. 271:3154-3162, 1996.

Zvegf3 proteins are purified by conventional protein purificationmethods, typically by a combination of chromatographic techniques. See,in general, Affinity Chromatography: Principles& Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York, 1994. Proteinscomprising a polyhistidine affinity tag (typically about 6 histidineresidues) are purified by affinity chromatography on a nickel chelateresin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325,1988. Furthermore, the growth factor domain itself binds to nickel resinat pH 7.0-8.0 and 25 mM Na phosphate, 0.25 M NaCl. Bound protein can beeluted with a descending pH gradient down to pH 5.0 or an imidazolegradient. Proteins comprising a glu-glu tag can be purified byimmunoaffinity chromatography according to conventional procedures. See,for example, Grussenmeyer et al., ibid. Maltose binding protein fusionsare purified on an amylose column according to methods known in the art.As disclosed in more detail below, zvegf3 growth factor domain proteincan be purified using a combination of chromatography on a strong cationexchanger followed by hydrophobic interaction chromatography. When theprotein is produced in BHK cells, insulin-like growth factor bindingprotein 4 (IGFBP4) co-purifies with the zvegf3 under these conditions.Further purification can be obtained using reverse-phase HPLC, anionexchange on a quaternary amine strong cation exchanger at low ionicstrength and pH from 7.0 to 9.0, or hydrophobic interactionchromatography on a phenyl ether resin. It has also been found thatzvegf3 binds to various dye matrices (e.g., BLUE1, BLUE 2, ORANGE 1,ORANGE 3, and RED3 from Lexton Scientific, Signal Hill, Calif.) in PBSat pH 6-8, from which the bound protein can be eluted in 1-2M NaCl in 20mM boric acid buffer at pH 8.8. Protein eluted from RED3 may be passedover RED2 (Lexton Scientific) to remove remaining contaminants.

Zvegf3 proteins can be used wherever it is desired to stimulate theproduction of bone and/or connective tissue in both humans and non-humananimals. Veterinary uses include use in domestic animals, includinglivestock and companion animals. Specific applications include, withoutlimitation, fractures, including non-union fractures and fractures inpatients with compromised healing, such as diabetics, alcoholics, andthe aged; bone grafts; healing bone following radiation-inducedosteonecrosis; implants, including joint replacements and dentalimplants; repair of bony defects arising from surgery, such ascranio-maxilofacial repair following tumor removal, surgicalreconstruction following traumatic injury, repair of hereditary or otherphysical abnormalities, and promotion of bone healing in plasticsurgery; treatment of periodontal disease and repair of other dentaldefects; treatment of bone defects following therapeutic treatment ofbone cancers; increase in bone formation during distractionosteogenesis; treatment of joint injuries, including repair of cartilageand ligament; repair of joints that have been afflicted withosteoarthritis; tendon repair and re-attachment; treatment ofosteoporosis (including age-related osteoporosis, post-menopausalosteoporosis, glutocorticoid-induced osteoporosis, and disuseosteoporosis) and other conditions characterized by increased bone lossor decreased bone formation; elevation of peak bone mass inpre-menopausal women; and use in the healing of connective tissuesassociated with dura mater.

For use within the present invention, zvegf3 proteins are formulated forlocal or systemic (particularly intravenous or subcutaneous) deliveryaccording to conventional methods. In general, pharmaceuticalformulations will include a zvegf3 protein in combination with apharmaceutically acceptable delivery vehicle. Delivery vehicles includebiocompatible solid or semi-solid matrices, including powdered bone,ceramics, biodegradable and non-biodegradable synthetic polymers, andnatural polymers; tissue adhesives (e.g., fibrin-based); aqueouspolymeric gels; aqueous solutions; liposomes; and the like. Exemplaryformulations and delivery vehicles are disclosed below. This disclosureis illustrative; those skilled in the art will readily recognizesuitable alternatives, including derivatives of the specifically namedmaterials and combinations of materials. Formulations may furtherinclude one or more additional growth factors, excipients,preservatives, solubilizers, buffering agents, albumin to preventprotein loss on vial surfaces, etc. Methods of formulation are wellknown in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. An “effective amount” of a composition isthat amount that produces a statistically significant effect, such as astatistically significant increase in the rate of fracture repair,reversal of bone loss in osteoporosis, increase in the rate of healingof a joint injury, increase in the reversal of cartilage defects,increase or acceleration of bone growth into prosthetic devices,improved repair of dental defects, and the like. The exact dose will bedetermined by the clinician according to accepted standards, taking intoaccount the nature and severity of the condition to be treated, patienttraits, etc. Determination of dose is within the level of ordinary skillin the art. Depending upon the route and method of administration, theprotein may be administered in a single dose, as a prolonged infusion,or intermittently over an extended period. Intravenous administrationwill be by bolus injection or infusion over a typical period of one toseveral hours. Sustained release formulations can be employed. Ingeneral, a therapeutically effective amount of zvegf3 is an amountsufficient to produce a clinically significant change in the treatedcondition, such as a clinically significant reduction in time requiredfor fracture repair, a significant reduction in the volume of a void orother defect, a significant increase in bone density, a significantreduction in morbidity, or a significantly increased histological score.

Zvegf3 will ordinarily be used in a concentration of about 10 to 100μg/ml of total volume, although concentrations in the range of 1 ng/mlto 1000 μg/ml may be used. For local application, such as for theregeneration of bone in a fracture or other bony defect, the proteinwill be applied in the range of 0.1-100 μg/cm² of wound area.

Within the present invention zvegf3 can be used in combination withother growth factors and other therapeutic agents that have a positiveeffect on the growth of bone or connective tissue. Such growth factorsinclude insulin-like growth factor 1 (IGF-1), PDGF, alpha and betatransforming growth factors (TGF-α and TGF-β), epidermal growth factor(EGF), bone morphogenetic proteins, leukemia inhibitory factor,fibroblast growth factors, and zvegf4 proteins (e.g., a dimeric proteincomprising two disulfide-bonded polypeptide chains, each of said chainscomprising residues 258-370 of SEQ ID NO:5). Other therapeutic agentsinclude vitamin D, bisphosphonates, calcitonin, estrogens, parathyroidhormone, osteogenin, NaF, osteoprotegerin, and statins.

Zvegf3 can be delivered as a component of a tissue adhesive.Fibrin-based tissue adhesives are known in the art, and can be preparedfrom plasma or recombinant sources. Tissue adhesives comprise fibrinogenand factor XIII to which thrombin is added immediately before use toactivate cross-linking. See, for example, Schwarz et al., U.S. Pat. No.4,414,976; Stroetmann et al., U.S. Pat. No. 4,427,650; and Rose et al.,U.S. Pat. No. 4,928,603. The use of tissue adhesives may be particularlyadvantageous in the treatment of conditions where connective tissue mustbe repaired, such as torn ligaments or tendons. Zvegf3 may also becombined with collagen-based adhesives. The collagen may be isolatedfrom natural or recombinant sources.

Solid and semisolid matrices are preferred delivery vehicles for fillingnon-union fractures, cavities, and other bony defects. These matricesprovide a space-filling substitute for the natural bone, and includebone substituting agents such as tricalcium phosphate, hydroxyapatite,combinations of tricalcium phosphate and hydroxyapatite,polymethylmethacrylate, aluminates and other ceramics, and demineralizedfreeze-dried cortical bone. Solid and semi-solid matrices can also beprepared from a variety of polymeric materials. Semi-solid matricesprovide the advantage of maleability such that they can be shaped toprovide a precise filling of a bony defect. Matrices may include otheractive or inert components. Of particular interest are those agents thatpromote tissue growth or infiltration. Agents that promote bone growthinclude bone morphogenic proteins (U.S. Pat. No. 4,761,471; PCTPublication WO 90/11366), osteogenin (Sampath et al., Proc. Natl. Acad.Sci. USA 84: 7109-7113, 1987), and NaF (Tencer et al., J. Biomed. Mat.Res. 23: 571-589, 1989).

Biodegradable, synthetic polymers include polyesters, polyorthoesters,polyanhydrides, polycarbonates, polyfumarates, polyhydroxybutyrate,vinyl polymers, and the like. Specific examples include, withoutlimitation, polylactide, polyglycolide, polylactide/polyglycolidecopolymers, polydioxanone, polyglycolide/trimethylene carbonatecopolymers, polyacrylic acid, polymethacrylic acid, polyvinylpyrrolidone, and polyvinyl alcohol. Such materials can be prepared in avariety shapes, including films, plates, pins, rods, screws, blocks,lattices, and the like for attachment to or insertion into bone. See,for example, Walter et al., U.S. Pat. No. 5,863,297; and WIPOpublication WO 93/20859. These materials may further include a carriersuch as albumin, a polyoxyethylenesorbitan detergent or glutamic acid.In principle, any substance that enhances polymer degradation, createspores in the matrix or reduces adsorption of the growth factor(s) to thematrix can be used as a carrier. Polyoxyethylenesorbitan detergents thatare useful as carriers include polyoxyethylenesorbitan monooleate,polyoxyethylenesorbitan monolaureate, polyoxyethylenesorbitanmonopalmitate, polyoxy-ethylenesorbitan monostearate andpolyoxyethylenesorbitan trioleate. Plasticizers can also be included.

In general, a film or device as described herein is applied to the boneat a site of injury. Application is generally by implantation into thebone or attachment to the surface using standard surgical procedures.

Biodegradable polymer films are particularly useful as coatings forprosthetic devices and surgical implants. Such films can, for example,be wrapped around the outer surfaces of surgical screws, rods, pins,plates and the like, or can themselves be rolled or otherwise formedinto a variety of shapes. Implantable devices of this type are routinelyused in orthopedic surgery. Films can also be used to coat bone fillingmaterials, such as hydroxyapatite blocks, demineralized bone matrixplugs, collagen matrices, and the like.

As used herein the term “copolymer” includes any polymer containing twoor more types of monomer unit. Copolymers can be classified in fourtypes as shown in the following chart, wherein “A” and “B” denote thecomponent monomer units:

Degradation of the matrix and consequent release of growth factorstherefrom can be modulated by adjusting such parameters as molecularweight, copolymer structure, copolymer ratio, matrix thickness, andporosity, and by including a carrier as disclosed above. PLA/PGA films,for example, are generally formulated to provide a ratio of PLA:PGAbetween 75:25 and 25:75, more commonly between 65:35 and 35:65. Ingeneral, an implant will be prepared using a copolymer having amolecular weight between 10,000 and 200,000 Daltons. In general, lowermolecular weight copolymers will degrade more rapidly than highermolecular weight formulations; random copolymers are less crystallineand therefore degrade more quickly than other types of copolymers; andpolymers of enantiomeric lactides are crystalline and therefore moreresistant to degradation than their racemic counterparts.

Polymer matrices are prepared according to procedures known in the art.See, for example, Loomis et al., U.S. Pat. No. 4,902,515; Gilding andReed, Polymer 20: 1459-1464, 1979; and Boswell et al., U.S. Pat. No.3,773,919. For example, PLA/PGA copolymer implants are produced bycombining the desired amount of PLA/PGA copolymer granules in a suitablesolvent (e.g., chloroform or methylene chloride), pouring the resultingsolution into a mold, and completely evaporating the solvent. In thealternative, PLA/PGA implants can be produced by compression molding,extrusion, or other known methods. To load the matrix, zvegf3 and acarrier are applied as powders or liquid solutions. For example,lyophilized zvegf3 and albumin may be uniformly dispersed over onesurface of polymer film, and the film folded over. By repeated thisprocess, a multi-layered “sandwich” of polymer and growth factor can beconstructed. In the alternative, the proteins can be applied as aqueoussolutions (e.g., in phosphate buffered saline or 0.1 M acetic acid),which are allowed to dry. Porous implants can be soaked in a solution ofzvegf3 (optionally containing other components), and the liquidevaporated. Zvegf3 can be worked into a maleable polymeric matrix afterwhich the matrix is formed into the desired shape and cured at elevatedtemperature (e.g., 60-65° C.). Porous implants can be prepared by curingthe matrix under vacuum.

Zvegf3 can also be delivered in combination with a biodegradable sponge,for example a gelatin, collagen, cellulose, or chitin sponge. Suchsponges are known in the art. See, for example, Correll, U.S. Pat. No.2,465,357; Miyata et al., U.S. Pat. No. 4,271,070; and Munck et al., WO90/13320. A solution of zvegf3 and, optionally, one or more additionaltherapeutic agents is injected into the sponge, and the sponge isair-dried at a temperature of 30-100° C. for a time sufficient to reducethe water content to below 50%, preferably below 10%.

Gels can also be used as delivery vehicles. The use of aqueous,polymeric gels for the delivery of growth factors is disclosed by, forexample, Finkenaur et al., U.S. Pat. No. 5,427,778; Edwards et al., U.S.Pat. No. 5,770,228; and Finkenaur et al., U.S. Pat. No. 4,717,717; andCini et al., U.S. Pat. No. 5,457,093. Gels comprise biocompatible, watersoluble or water swellable polymers that form viscous solutions inwater. Such polymers include, without limitation, polysaccharides,including methyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxyethyl cellulose, dextrans, starch, chitosan, andalginic acid; glycosaminoglycans, including hyaluronic acid,chondroitin, chondroitin sulfates, heparin, and heparan sulfate;proteins, including collagen, gelatin, and fibronectin; and acrylamides,including polyacrylamide and polymethacrylamide. Gels are generallyprepared with a viscosity of from 200 cps to 100,000 cps, more commonlyabout 1000 cps to 30,000 cps at room temperature, the latter rangecorresponding to about 0.25-10% hydroxyethyl cellulose in water. Higherviscosity gels are known in the art (e.g., Finkenaur et al., U.S. Pat.No. 5,427,778). Viscosity can be adjusted by varying the concentrationand/or length of the component polymer(s). Gels are prepared bycombining the polymer with a suitable buffer, such as a low ionicstrength citrate, phosphate, or acetate buffer at neutral or slightlyacidic pH. A preservative (antimicrobial agent) such as methyl paraben,propyl paraben, benzyl alcohol, or the like, will generally be included.Following thorough mixing, the solution is sterilized by suitable means(e.g., autoclaving). The mixture is cooled, and filter-sterilized zvegf3is added.

Alternative means for local delivery of zvegf3 include osmotic minipumps(e.g., ALZET® minipumps; Alza Corporation, Mountain View, Calif.);electrically charged dextran beads as disclosed in Bao et al. (WO92/03125); collagen-based delivery systems, such as disclosed in Ksanderet al. (Ann. Surg. 211:288-294, 1990); and alginate-based systems asdisclosed in Edelman et al. (Biomaterials, 12:619-626, 1991). Othermethods known in the art for sustained local delivery in bone includeporous coated metal protheses that can be impregnated with a therapeuticagent and solid plastic rods with therapeutic compositions incorporatedwithin them.

Zvegf3 can be further used to treat osteoporosis by administering atherapeutically effective amount of zvegf3 to an individual. Zvegf3proteins can be tested in intact animals using an in vivo dosing assay.Prototypical dosing may be accomplished by subcutaneous, intraperitonealor oral administration, and may be performed by injection, sustainedrelease or other delivery techniques. The time period for administrationof zvegf3 may vary (for instance, 28 days as well as 35 days may beappropriate).

Delivery of systemically adminstered compositions of the presentinvention may be enhanced by conjugating zvegf3 to a targeting molecule.A “targeting molecule” is a molecule that binds to the tissue ofinterest. For example, bone-targeting molecules include tetracyclines,calcein, bisphosphonates, polyaspartic acid, polyglutamic acid,aminophosphosugars, peptides known to be associated with the mineralphase of bone (e.g., osteonectin, bone sialoprotein, and osteopontin),bone-specific antibodies, proteins with bone mineral or bone cellbinding domains (e.g., calcitonin), and the like. See, for example, thedisclosures of Bentz et al., EP 512,844; Murakami et al., EP 341,961;and Brinkley, Bioconjugate Chem. 3:2-13, 1992. Conjugation willordinarily be achieved through a covalent linkage, the precise nature ofwhich will be determined by the targeting molecule and the linking siteon the zvegf3 polypeptide. Typically, a non-peptidic agent is modifiedby the addition of a linker that allows conjugation to zvegf3 throughits amino acid side chains, carbohydrate chains, or reactive groupsintroduced on zvegf3 by chemical modification. For example, a drug maybe attached through the ε-amino group of a lysine residue, through afree α-amino group, by disulfide exchange to a cysteine residue, or byoxidation of the 1,2-diols in a carbohydrate chain with periodic acid toallow attachment of drugs containing various nucleophiles through aSchiff-base linkage. See, for example, Ali et al., U.S. Pat. No.4,256,833. Protein modifying agents include amine-reactive reagents(e.g., reactive esters, isothiocyantates, aldehydes, and sulfonylhalides), thiol-reactive reagents (e.g., haloacetyl derivatives andmaleimides), and carboxylic acid- and aldehyde-reactive reagents. Zvegf3polypeptides can be covalently joined to peptidic agents through the useof bifunctional cross-linking reagents. Heterobifunctional reagents aremore commonly used and permit the controlled coupling of two differentproteins through the use of two different reactive moieties (e.g.,amine-reactive plus thiol, iodoacetamide, or maleimide). The use of suchlinking agents is well known in the art. See, for example, Brinkley(ibid.) and Rodwell et al., U.S. Pat. No. 4,671,958. Peptidic linkerscan also be employed. In the alternative, a zvegf3 polypeptide can belinked to a peptidic moiety through preparation of a fusion polypeptide.

Zvegf3 can be implanted in a mammalian body so that the zvegf3 is incontact with osteoblasts such that osteoblast proliferation occurs andbone growth is stimulated. For example, zvegf3 can be placed in a matrixin association with a bone morphogenic protein (BMP). The BMP inducesthe migration of mesenchymal osteoblast precursors to the site andfurther induces differentiation of the mesenchymal cells intoosteoblasts. Zvegf3 will then stimulate the further proliferation of theosteoblasts. A suitable matrix is made up of particles of porousmaterials. The pores must be of a dimension to permit progenitor cellmigration and subsequent differentiation and proliferation, generally inthe range of 70-850 μm, commonly from 150 μm to 420 μm. The matrixcontaining the zvegf3 can be molded into a shape encompassing a bonedefect. Examples of matrix materials are particulate, demineralized,guanidine extracted, species-specific bone. Other potentially usefulmatrix materials include collagen, homopolymers and copolymers ofglycolic acid and lactic acid, hydroxyapatite, tricalcium phosphate andother calcium phosphates. Zvegf3 can be applied into a matrix at asufficient concentration to promote the proliferation of osteoblasts,preferably at a concentration of at least 1 μg/ml of matrix. A solutionof zvegf3 can also be injected directly into the site of a bone fractureto expedite healing of the fracture. Examples of BMPs and the use ofmatrices to produce are disclosed in PCT application publication numberWO 92/07073, publication No. WO 91/05802,U.S. Pat. Nos. 5,645,591 and5,108,753.

As stated above, it has also been determined that zvegf3 can be used topromote the production of cartilage through its ability to stimulate theproliferation of chondrocytes. Zvegf3 can be injected directly into thesite where cartilage is to be grown. For example, zvegf3 can be injecteddirectly in joints which have been afflicted with osteoarthritis orother injured joints in which the cartilage has been worn down ordamaged by trauma. In the alternative, zvegf3 can be delivered in asuitable solid or semi-solid matrix as disclosed above.

Cartilage can also be grown by first removing chondrocytes from apatient and culturing them in the presence of zvegf3 so that theyproliferate. Chondrocytes are cultured for from several hours to a dayor longer according to conventional methods in a culture medium (e.g.,DMEM supplemented with 10% patient's serum) containing from about 0.01μg/ml to about 100 μg/ml zvegf3. The proliferated chondrocytes arereimplanted into the patient where cartilage needs to be produced. Theproliferated chondrocytes can be delivered in a porous matrix havingsufficient porosity to permit cell ingrowth as generally disclosedabove. Additional zvegf3 can be included in the matrix to promotefurther chondrocyte proliferation after implantation. See, in general,Walter et al., U.S. Pat. No. 5,863,297 and Boyan et al., U.S. Pat. No.6,001,352.

Within another embodiment, the present invention provides methods forstimulating the growth and/or differentiation of bone-forming andcartilage-forming cells, or their precursors, in vitro. Using thesemethods, cells can be harvested from a patient, expanded ex vivo, andreturned to the patient as generally disclosed above. Of particularinterest is the growth and/or differentiation of bone marrow cells,which can be cultured in the presence of differentiation-stimulatingagents to develop into, inter alia, osteoblasts, osteoclasts, andchondrocytes. Identification of differentiated cells within a primaryculture is primarily phenotypic. For example, the phenotypic markers forosteoblasts include expression of alkaline phosphatase (Manduca et al.,J. Bone Min. Res. 8:281, 1993), type 1 collagen synthesis (Kurihara etal., Endocrinol. 118(3):940-947, 1986), production of osteocalcin (Yoonet al., Biochem. 27:8521-8526, 1988) and responsiveness to parathyroidhormone (Aubin et al., J. Cell Biol, 92:452-461, 1982). Osteoblast cellsare typically cultured at 37° C. in 5% CO₂ in a growth medium thatincludes a carbon source, a nitrogen source, essential amino acids,vitamins, minerals and growth factors generally supplied by fetal calfserum. A variety of suitable media are known in the art. Zvegf3polypeptides are added to tissue culture media for these cell types at aconcentration of about 10 pg/ml to about 1000 ng/ml. Those skilled inthe art will recognize that zvegf3 proteins can be advantageouslycombined with other growth factors in culture media.

Bony defects or connective tissue injuries may also be repaired using agene therapy approach wherein a polynucleotide encoding zvegf3 isadministered to a patient. Gene delivery systems useful in this regardinclude adenovirus, adeno-associated virus, and naked DNA vectors. See,for example, by Anderson et al., U.S. Pat. No. 5,399,346; Mann et al.,Cell 33:153, 1983; Temin et al., U.S. Pat. No. 4,650,764; Temin et al.,U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol 62:1120, 1988; Teminet al., U.S. Pat. No. 5,124,263; Dougherty et al., WIPO publication WO95/07358; and Kuo et al., Blood 82:845, 1993. Of particular interest islocal infection of the affected tissue, such as local application of thevector to a periodontal pocket, fracture, joint, implant site, or siteof prosthetic attachment.

The invention is further illustrated by the following, non-limitingexamples.

EXAMPLE 1

An expression plasmid containing all or part of a polynucleotideencoding zvegf3 is constructed via homologous recombination. A fragmentof zvegf3 cDNA is isolated by PCR using the polynucleotide sequence ofSEQ ID NO: 1 with flanking regions at the 5′ and 3′ ends correspondingto the vector sequences flanking the zvegf3 insertion point. The primersfor PCR each include from 5′ to 3′ end: 40 bp of flanking sequence fromthe vector and 17 bp corresponding to the amino and carboxyl terminifrom the open reading frame of zvegf3.

Ten μl of the 100 μl PCR reaction is run on a 0.8%low-melting-temperature agarose (SEAPLAQUE GTG®; FMC BioProducts,Rockland, Me.) gel with 1×TBE buffer for analysis. The remaining 90 μlof PCR reaction is precipitated with the addition of 5 μl 1 M NaCl and250 μl of absolute ethanol. The plasmid pZMP6, which has been cut withSmaI, is used for recombination with the PCR fragment. Plasmid pZMP6 wasconstructed from pZP9 (deposited at the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, underAccession No. 98668) with the yeast genetic elements taken from pRS316(deposited at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, under Accession No. 77145), aninternal ribosome entry site (IRES) element from poliovirus, and theextracellular domain of CD8 truncated at the C-terminal end of thetransmembrane domain. pZMP6 is a mammalian expression vector containingan expression cassette having the cytomegalovirus immediate earlypromoter, multiple restriction sites for insertion of coding sequences,a stop codon, and a human growth hormone terminator. The plasmid alsocontains an E. coli origin of replication; a mammalian selectable markerexpression unit comprising an SV40 promoter, enhancer and origin ofreplication, a DHFR gene, and the SV40 terminator; as well as the URA3and CEN-ARS sequences required for selection and replication in S.cerevisiae.

One hundred microliters of competent yeast (S. cerevisiae) cells areindependently combined with 10 μl of the various DNA mixtures from aboveand transferred to a 0.2-cm electroporation cuvette. The yeast/DNAmixtures are electropulsed using power supply settings of 0.75 kV (5kV/cm), ∞ ohms, 25 μF. To each cuvette is added 600 μl of 1.2 Msorbitol, and the yeast is plated in two 300-μl aliquots onto two URA-Dplates and incubated at 30° C. After about 48 hours, the Ura⁺ yeasttransformants from a single plate are resuspended in 1 ml H₂O and spunbriefly to pellet the yeast cells. The cell pellet is resuspended in 1ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH8.0, 1 mM EDTA). Five hundred microliters of the lysis mixture is addedto an Eppendorf tube containing 300 μl acid-washed glass beads and 200μl phenol-chloroform, vortexed for 1 minute intervals two or threetimes, and spun for 5 minutes in an Eppendorf centrifuge at maximumspeed. Three hundred microliters of the aqueous phase is transferred toa fresh tube, and the DNA is precipitated with 600 μl ethanol (EtOH),followed by centrifugation for 10 minutes at 4° C. The DNA pellet isresuspended in 10 μl H₂O.

Transformation of electrocompetent E. coli host cells (ELECTROMAX DH10B™cells; obtained from Life Technologies, Inc., Gaithersburg, Md.) is donewith 0.5-2 ml yeast DNA prep and 40 ul of cells. The cells areelectropulsed at 1.7 kV, 25 μF, and 400 ohms. Following electroporation,1 ml SOC (2% BACTO™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) is plated in 250-μl aliquots on four LB AMP plates (LB broth(Lennox), 1.8% BACTO™ Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct expression construct for zvegf3are identified by restriction digest to verify the presence of thezvegf3 insert and to confirm that the various DNA sequences have beenjoined correctly to one another. The inserts of positive clones aresubjected to sequence analysis. Larger scale plasmid DNA is isolatedusing a commercially available kit (QIAGEN™ Plasmid Maxi Kit, Qiagen,Valencia, Calif.) according to manufacturer's instructions. The correctconstruct is designated pZMP6/zvegf3.

EXAMPLE 2

Full-length zvegf3 protein was produced in BHK cells transfected withpZMP6/zvegf3. BHK 570 cells (ATCC CRL-10314) were plated in 10-cm tissueculture dishes and allowed to grow to approximately 50 to 70% confluenceovernight at 37° C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL HighGlucose; Life Technologies), 5% fetal bovine serum (Hyclone, Logan,Utah), 1 mM L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mM sodiumpyruvate (Life Technologies). The cells were then transfected withpZMP6/zvegf3 by liposome-mediated transfection (using (LIPOFECTAMINE™;Life Technologies), in serum free (SF) media (DMEM supplemented with 10mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and1% sodium pyruvate). The plasmid was diluted into 15-ml tubes to a totalfinal volume of 640 μl with SF media. 35 μl of the lipid mixture wasmixed with 605 μl of SF medium, and the mixture was allowed to incubateapproximately 30 minutes at room temperature. Five milliliters of SFmedia was added to the DNA:lipid mixture. The cells were rinsed oncewith 5 ml of SF media, aspirated, and the DNA:lipid mixture was added.The cells were incubated at 37° C. for five hours, then 6.4 ml ofDMEM/10% FBS, 1% PSN media was added to each plate. The plates wereincubated at 37° C. overnight, and the DNA:lipid mixture was replacedwith fresh 5% FBS/DMEM media the next day. On day 5 post-transfection,the cells were split into T-162 flasks in selection medium (DMEM+5% FBS,1% L-Gln, 1% NaPyr, 1 μM methotrexate). Approximately 10 dayspost-transfection, two 150-mm culture dishes of methotrexate-resistantcolonies from each transfection were trypsinized, and the cells arepooled and plated into a T-162 flask and transferred to large-scaleculture.

EXAMPLE 3

Recombinant zvegf3 growth factor domain was produced in culturedmammalian cells. A mammalian cell expression vector for the growthfactor domain of zvegf3 was constructed essentially as disclosed inExample 1. The coding sequence for the growth factor domain (residues235-345 of SEQ ID NO:2), joined to a sequence encoding an optimized t-PAsecretory signal sequence (U.S. Pat. No. 5,641,655) was joined to thelinearized pZMP11 vector downstream of the CMV promoter. The plasmidpZMP11 is a mammalian expression vector containing an expressioncassette having the CMV immediate early promoter, a consensus intronfrom the variable region of mouse immunoglobulin heavy chain locus,Kozak sequences, multiple restriction sites for insertion of codingsequences, a stop codon, and a human growth hormone terminator. Theplasmid also contains an IRES element from poliovirus, the extracellulardomain of CD8 truncated at the C-terminal end of the transmembranedomain, an E. coli origin of replication, a mammalian selectable markerexpression unit having an SV40 promoter, enhancer and origin ofreplication, a DHFR gene, the SV40 terminator, and the URA3 and CEN-ARSsequences required for selection and replication in S. cerevisiae. Theresulting vector was designated pZMP11/zv3GF-otPA. BHK 570 cells weretransfected with pZMP11/zv3GF-otPA and cultured essentially as disclosedin Example 2.

EXAMPLE 4

Recombinant zvegf3 growth factor domain was produced in BHK 570 cellsgrown in cell factories. Three 15-liter cultures were harvested, and themedia were sterile filtered using a 0.2 μ filter. Expression levels wereestimated by western blot analysis of media samples concentrated to 20×using a 5K cut-off membrane and serially diluted by two-fold to 1.25×concentration. Signal intensity was compared to a signal on the sameblot from an MBP-zvegf3 fusion protein standard for which the proteinconcentration had been determined by amino acid analysis. Expressionlevels were consistently between 0.25 and 0.35 mg/L of media.

Protein was purified from conditioned media by a combination of cationexchange chromatography and hydrophobic interaction chromatography.Culture medium was diluted with 0.1 M acetic acid, pH 3.0, containing0.3 M NaCl at a ratio of 60%:40%, (medium:acetic acid) to deliver aprocess stream at 14 mS conductivity and pH 4.0. This stream wasdelivered to a strong cation exchange resin (POROS® HS; PerSeptiveBiosystems, Framingham, Mass.) with a bed volume of 50 ml in a 2-cmdiameter column at a flow rate of 20 ml/minute. A 50-ml bed wassufficient to process 45 L of media and capture all of the targetprotein. Bound protein was eluted, following column washing for 10column volumes in 10 mM acetic acid with 0.15 M NaCl at pH=4.0, byforming a linear gradient to 2M NaCl in 10 mM acetic acid, pH 4.0.Ten-ml fractions were captured into tubes containing 2 ml 2.0 M Tris, pH8.0 to neutralize the acidity. Samples from the cation exchange columnwere analyzed by SDS PAGE with silver staining and western blotting forthe presence of zvegf3. The vegf3 growth domain eluted at 0.2-0.5 MNaCl. Protein-containing fractions were pooled. A 25-ml bed ofchromatography medium (Toso Haas Ether chromatography medium) in a 2 cmdiameter column was equilibrated in 1.8 M (NH₄)₂SO₄ in 25 mM Naphosphate buffer at pH 7.4. The pooled protein from the cation exchangestep was adjusted to 1.8 M (NH₄)₂SO₄ in 25 mM Na phosphate, pH 7.0. Thisstream was flowed over the column at 10 ml/minute. Once the loading wascompleted the column was washed for 10 column volumes with theequilibration buffer prior to eluting with a 10 column volume gradientformed between the equilibration buffer and 40 mM boric acid at pH 8.8.The zvegf3 growth factor domain protein eluted fairly early in thegradient between 1.5 and 1.0 M (NH₄)₂SO₄. At this point the protein was40-60% pure with a major contaminant being insulin-like growth factorbinding protein 4 (IGFBP4).

Protein from the HIC (Ether) chromatography step was applied to a C4reverse-phase HPLC column. The zvegf3 growth factor domain proteineluted at 36% acetonitrile. This material still contained approximately20% (mole/mole) IGFB4.

EXAMPLE 5

Recombinant zvegf3 growth factor domain is purified fromcell-conditioned media by a combination of cation exchangechromatography, hydrophobic interaction chromatography, and nickelaffinity chromatography. Protein is captured on a strong cation exchangemedium and eluted essentially as disclosed in Example 4. The elutedprotein is further purified by hydrophobic interaction chromatography onan ether resin (POROS® ET; PerSeptive Biosystems). The partiallypurified zvegf3 protein is then bound to a nickel chelate resin at pH7.0-8.0 in 25 mM Na phosphate buffer containing 0.25 M NaCl. The boundprotein is eluted with a descending pH gradient down to pH 5.0 or animidazole gradient. The eluate from the nickel column is adjusted to 1 M(NH₄)₂SO₄, 20 mM MES (morphilino ethanesulfonic acid) at pH 6.0 andpassed through a phenyl ether hydrophobic interaction chromatographycolumn (POROS® PE, PerSeptive Biosystems) that has been equilibrated in1 M (NH₄)₂SO₄, 20 mM MES, pH 6.0. IGFBP4 and minor contaminants areretained on the column. The pass-through fraction, which contains highlypurified zvegf3, is collected. The collected protein is desaltedaccording to conventional methods (e.g., dialysis, ion-exchangechromatography).

EXAMPLE 6

Recombinant zvegf3 was analyzed for mitogenic activity on human aorticsmooth muscle cells (HAoSMC; Clonetics Corp., Walkersville, Md.) andhuman umbilical vein endothelial cells (HUVEC; Clonetics Corp.). HAoSMCand HUVEC were plated at a density of 5,000 cells/well in 96-wellculture plates and grown for approximately 24 hours in DMEM containing10% fetal calf serum at 37° C. Cells were quiesced by incubating themfor 24 hours in serum-free DMEM/Ham's F-12 medium containing insulin (5μg/ml), transferrin (20 μg/ml), and selenium (16 pg/ml) (ITS). At thetime of the assay, the medium was removed, and test samples were addedto the wells in triplicate. Test samples consisted of either conditionedmedia (CM) from adenovirally-infected HaCaT human keratinocyte cells(Boukamp et al., J. Cell Biol. 106:761-771, 1988) expressing full-lengthzvegf3, purified growth factor domain expressed in BHK cells, or controlmedia from cells infected with parental adenovirus (Zpar). The CM wasconcentrated 10-fold using a 15 ml centrifugal filter device with a 10Kmembrane filter (ULTRAFREE®; Millipore Corp., Bedford, Mass.), thendiluted back to 3× with ITS medium and added to the cells. The controlCM was generated from HaCaT cells infected with a parental greenfluorescent protein-expressing adenovirus and treated identically to thezvegf3 CM. Purified protein in a buffer containing 0.1% BSA was seriallydiluted into ITS medium at concentrations of 1 μg/ml to 1 ng/ml andadded to the test plate. A control buffer of 0.1% BSA was dilutedidentically to the highest concentration of zvegf3 protein and added tothe plate. For measurement of [³H]thymidine incorporation, 20 μl of a 50μCi/ml stock in DMEM was added directly to the cells, for a finalactivity of 1 μCi/well. After another 24 hour incubation, mitogenicactivity was assessed by measuring the uptake of [³H]thymidine. Mediawere removed and cells were incubated with 0.1 ml of trypsin until cellsdetached. Cells were harvested onto 96-well filter plates using a sampleharvester (FILTERMATE™ harvester; Packard Instrument Co., Meriden,Conn.). The plates were then dried at 65° C. for 15 minutes, sealedafter adding 40 μl/well scintillation cocktail (MICROSCINT™ O; PackardInstrument Co.) and counted on a microplate scintillation counter(TOPCOUNT®; Packard Instrument Co.).

Results presented in Table 2 demonstrate that zvegf3 CM hadapproximately 1.5-fold higher mitogenic activity on HAoSM cells overcontrol CM, and purified protein caused a maximal 1.8-fold increase in[³H]thymidine incorporation over the buffer control.

TABLE 2 CPM Incorporated Sample Mean St. dev. zvegf3 (3× CM) 81089 8866Zpar (3× CM) 58760 2558 zvegf3 GF domain, 1 μg/ml 63884 3281 zvegf3 GFdomain, 500 ng/ml 57484 9744 zvegf3 GF domain, 100 ng/ml 70844 10844zvegf3 GF domain, 50 ng/ml 61164 2813 zvegf3 GF domain, 10 ng/ml 606761514 zvegf3 GF domain, 5 ng/ml 60197 2481 zvegf3 GF domain, 1 ng/ml49205 5208 Buffer control 39645 9793 PDGF 10 ng/ml (maximal response)50634 4238 Media alone (basal response) 24220 2463

Results presented in Table 3 demonstrate that zvegf3 CM had no mitogenicactivity on HUVEC compared to the control CM, and purified proteincaused a maximal 1.3-fold increase in [³H]thymidine incorporation overthe buffer control.

TABLE 3 CPM Incorporated Sample Mean St. dev. zvegf3 (3× CM) 62723 10716Zpar (3× CM) 61378 1553 zvegf3 VEGF domain, 1 μg/ml 44901 6592 zvegf3VEGF domain, 500 ng/ml 41921 5330 zvegf3 VEGF domain, 100 ng/ml 356135187 zvegf3 VEGF domain, 50 ng/ml 31107 525 zvegf3 VEGF domain, 10 ng/ml28505 2950 zvegf3 VEGF domain, 5 ng/ml 29290 988 zvegf3 VEGF domain, 1ng/ml 28586 2718 Buffer control 33461 404 VEGF 50 ng/ml (maximalresponse) 53225 5229 Media alone (basal response) 22264 2814

EXAMPLE 7

Recombinant zvegf3 protein was assayed for stimulation of intracellularcalcium release as an indicator of receptor binding and activation.Cells were cultured in chambered borosilicate coverglass slides. On theday of assay, cells were incubated for 30 minutes at room temperature inKRW buffer (KrebsRingerWollheim; 140 mM NaCl, 3.6 mM KCl, 0.5 mMNaH₂PO₄, 0.5 mM MgSO₄ 2 mM NaHCO₃, 3 mM glucose, 1.5 mM CaCl₂, 10 mMHEPES pH 7.4) containing 2 μM fura-2 AM (obtained from Molecular ProbesInc., Eugene, Oreg.), washed twice with KRW buffer, and allowed to sitat room temperature for at least 15 minutes before addition of growthfactor or cell-conditioned culture medium (CM) to be tested. Changes incytosolic calcium were measured by fluorescence ratio imaging(excitation at 340 nm divided by excitation at 380 nm). Digital imagingwas carried out using an inverted fluorescent microscope (Nikon TE300)equipped with an oil objective (Nikon 40× Plan Fluor). Images wereacquired using a Princeton CCD digital camera and analyzed withUniversal Imaging Metafluor software. Data are presented in Table 4.

TABLE 4 Zvegf3 Control PDGF Cell Line CM CM VEGF BB aortic ring cells +− − + pericytes + − − + aortic smooth muscle cells + − − + aorticadventitial fibroblasts + − − +

EXAMPLE 8

Binding of recombinant zvegf3 to PDGF alpha and beta receptors wasmeasured by mass spectrometry using a surface-enhanced laser desorptionand ionization (SELDI) instrument (PROTEINCHIP™, Ciphergen Biosystems,Palo Alto, Calif.). For this experiment an 8-spot, preactivated surfacearray was used. To this amine-activated chip, protein-A (ZymedLaboratories, Inc., San Francisco, Calif.) was added at a concentrationof 1 mg/ml, and the chip was incubated at 4° C. for four hours. Afterblocking with 1M ethanolamine pH 8.0 and subsequent washes (once in 0.1%TRITON™ X-100 in PBS; once in 100 mM Na Acetate, pH4.5, 0.5 M NaCl; oncein 100 mM Tris-HCl, pH8.5, 0.5 M NaCl; once in PBS), IgG Fc-receptorextracellular domain fusion proteins (PDGF alpha receptor, PDGF betareceptor, or unrelated control receptor) were added, and the chip wasincubated at 4° C. overnight. After three washes in PBS, 250 μl ofzvegf3 (300 ng/ml), PDGF-AA, or PDGF-BB was added, and the chip wasincubated overnight at 4° C. The chip was washed twice with 0.05% TritonX100, 100 mM HEPES pH 7.2, then twice with deionized water. The chip wasallowed to dry at room temperature before two additions of 0.3microliters of sinapinic acid (Ciphergen Biosystems) in a 50:50 mixtureof acetonitrile and 1% trifluroacetic acid. Ligands that bound receptorwere retained on the chip after washing and subsequently detected bymass spectrometry. Assignment of a + or − for binding was made bycomparing the PDGF receptor mass spectrometry profile to that of an Fconly control for each ligand. Data are shown in Table 5.

TABLE 5 PDGF PDGF PDGF AA AB BB ZVEGF3 PDGFR-alpha/Fc + + + +PDGFR-beta/Fc +/− +/− + −

EXAMPLE 9

Hydroxyethyl cellulose (HEC; dry powder) is reconstituted in 100 mMsodium acetate buffer, pH 6.0 containing 0.2% (w/v) methyl paraben togive a concentration of 1.5% HEC (w/v). The mixture is sterilized byautoclaving at 120° for 20 minutes. Zvegf3 protein is added to a finalconcentration of 250 μg per gram of gel.

EXAMPLE 10

A 2.5% (w/v) hydroxypropylmethyl cellulose (HPMC) gel is prepared bydissolving powdered HPMC in 100 mM citrate buffer, pH 6.0 containing0.1% (w/v) methyl paraben. The mixture is sterilized by autoclaving at120° for 20 minutes. Zvegf3 protein is added to a final concentration of500 μg per gram of gel.

EXAMPLE 11

Zvegf3 is used to regenerate bone and ligament lost to periodontaldisease. Teeth showing 20% to 80% reduction of surrounding jaw bone arescaled, then a full-thickness gingival flap is made by an incision toexpose the jaw bone and tooth root. The root is planed to removebacterial plaque and calculus. Zvegf3 is applied to the periodontalpocket in a 2.5% HPMC gel at a dose of 100 μg per tooth. The gingivalflap is then closed and held in place by suturing.

EXAMPLE 12

For regeneration of bone lost to periodontal disease, affected teeth arescaled, and a full-thickness gingival flap is made by incision, exposingthe jaw bone and tooth root. The root is planed to remove bacterialplaque and calculus. A solution of zvegf3 in 100 mM sodium acetatebuffer, pH 6.0 is added to powdered bone to provide a dosage of 100 μgzvegf3 per tooth. The material is thoroughly mixed and applied to theexposed periodontal pocket. The gingival flap is then closed and held inplace by suturing.

EXAMPLE 13

Polylactic acid-polyglycolic acid films (50:50) are solvent cast bydissolving approximately 340 mg of polymer granules (MedisorbTechnologies International L.P, Wilmington, Del. or Polysciences,Warrington, Pa.) in 10 ml chloroform at room temperature and allowingthe solvent to evaporate completely in a slow air flow hood at roomtemperature. The films are approximately 10 μm thick. Each is cut into aca. 80 mm×40 mm sheet, resulting in a remaining polymer mass of about270-290 mg. A solution of zvegf3 and rabbit serum albumin is dispersedon the films, and the liquid is allowed to evaporate. The films are thenrolled around 0.9 mm diameter Kirschner wires (K-wires) to provideimplants of 1.5 or 3.0 mm diameter as shown in Table 6 and sterilizedusing cold ethylene oxide gas.

TABLE 6 Implant Diameter Zvegf3 (μg) Albumin (mg) 1.5 mm 100 40 3.0 mm10 40 3.0 mm 100 40

EXAMPLE 14

To test zvegf3 in an animal model of bone remodeling, seventythree-month-old female Sprague-Dawley rats are weight-matched anddivided into seven groups, with ten animals in each group. The studyincludes a baseline control group of animals sacrificed at theinitiation of the study, a control group administered vehicle only, aPBS-treated control group, and a positive control group administered acompound (non-protein or protein) known to promote bone growth. Threedosage levels of zvegf3 protein are administered to the remaining threegroups.

Briefly, zvegf3 protein, positive control compound, PBS, or vehiclealone is administered subcutaneously once per day for 35 days. Allanimals are injected with calcein nine days and two days beforesacrifice (two injections of calcein administered each designated day).Weekly body weights are determined. At the end of the 35-day cycle, theanimals are weighed and bled by orbital or cardiac puncture. Serumcalcium, phosphate, osteocalcin, and CBCs are determined. Both leg bones(femur and tibia) and lumbar vertebrae are removed, cleaned of adheringsoft tissue, and stored in 70% ethanol for evaluation. The effect ofzvegf3 protein on bone remodeling is performed by peripheralquantitative computed tomography (pQCT; Ferretti, Bone 17:353S-364S,1995), dual energy X-ray absorptiometry (DEXA; Laval-Jeantet et al.,Calcif Tissue Intl. 56:14-18, 1995; Casez et al, Bone and Mineral26:61-68, 1994) and/or histomorphometry.

EXAMPLE 15

Zvegf3 is tested in acute ovariectomized animals (prevention model)using an in vivo dosing assay with an estrogen-treated group as acontrol. Eighty three-month-old female Sprague-Dawley rats areweight-matched and divided into eight groups, with ten animals in eachgroup. This includes a baseline control group of animals sacrificed atthe initiation of the study; three control groups (sham ovariectomized(sham OVX)+vehicle only; ovariectomized (OVX)+vehicle only; PBS-treatedOVX); and a control OVX group that is administered estrogen. Threedosage levels of zvegf3 protein are administered to the remaining threegroups of OVX animals.

Since ovariectomy (OVX) induces hyperphagia, all OVX animals arepair-fed with sham OVX animals throughout the 35 day study. Briefly,test compound, positive control compound, PBS, or vehicle alone isadministered subcutaneously once per day for 35 days. Alternatively,test compound is formulated in implantable pellets that are implantedfor 35 days, or may be administered orally, such as by gastric gavage.All animals, including sham OVX/vehicle and OVX/vehicle groups, areinjected intraperitoneally with calcein nine days and two days beforesacrifice (two injections of calcein administered each designated day,to ensure proper labeling of newly formed bone). Weekly body weights aredetermined. At the end of the 35-day cycle, the animals' blood andtissues are processed as described above.

EXAMPLE 16

Zvegf3 is tested in chronic OVX animals (treatment model). 80 to 100six-month-old female, Sprague-Dawley rats are subjected to sham surgery(sham OVX) or ovariectomy (OVX) at time 0, and 10 rats are sacrificed toserve as baseline controls. Body weights are recorded weekly during theexperiment. After approximately 6 weeks of bone depletion (42 days), 10sham OVX and 10 OVX rats are randomly selected for sacrifice asdepletion period controls. Of the remaining animals, 10 sham OVX and 10OVX rats are used as placebo-treated controls. The remaining OVX animalsare treated with 3 to 5 doses of zvegf3 protein for a period of 5 weeks(35 days). As a positive control, a group of OVX rats is treated with anagent such as PTH, a known anabolic agent in this model (Kimmel et alEndocrinology 132:1577-1584, 1993). To determine effects on boneformation, the femurs, tibiae and lumbar vertebrae 1 to 4 are excisedand collected. The proximal left and right tibiae are used for pQCTmeasurements, cancellous bone mineral density (BMD) (gravimetricdetermination), and histology, while the midshaft of each tibiae issubjected to cortical BMD or histology. The femurs are prepared for pQCTscanning of the midshaft prior to biomechanical testing. With respect tolumbar vertebrae (LV), LV2 are processed for BMD (pQCT may also beperformed); LV3 are prepared for undecalcified bone histology; and LV4are processed for mechanical testing.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for promoting growth of bone, ligament, or cartilage in amammal comprising administering to said mammal a composition comprising:a pharmacologically effective amount of a dimeric protein comprising afirst polypeptide chain disulfide bonded to a second polypeptide chain,each of said chains consisting of an amino acid sequence having at least95% sequence identity with residues 235-345 of SEQ ID NO:2, wherein saiddimeric protein is capable of binding PDGF receptor α; and apharmaceutically acceptable delivery vehicle.
 2. The method of claim 1wherein the delivery vehicle is powdered bone, tricalcium phosphate,hydroxyapatite, polymethacrylate, a biodegradable polyester, an aqueouspolymeric gel, or a fibrin sealant.
 3. The method of claim 1 wherein thecomposition is locally administered at a site of a bony defect.
 4. Themethod of claim 3 wherein the bony defect is a fracture, bone graftsite, implant site, or periodontal pocket.
 5. The method of claim 1wherein the composition is administered systemically.
 6. The method ofclaim 1 wherein the composition is locally administered at a joint. 7.The method of claim 1 wherein the dimeric protein is covalently linkedto a bone-targeting agent.
 8. The method of claim 1 wherein thecomposition further comprises a protein selected from the groupconsisting of insulin-like growth factor 1, platelet-derived growthfactor, epidermal growth factor, transforming growth factor-alpha,transforming growth factor-beta, a bone morphogenetic protein,parathyroid hormone, osteoprotegerin, a fibroblast growth factor, and aprotein comprising residues 258-370 of SEQ ID NO:5.